Pseudouridine, the 5'ribosyl isomer of uridine, is the most common modified ribonucleoside and is found in all stable RNAs. Pseudouridine formation occurs posttranscriptionally, and its formation is catalyzed by pseudouridine synthases. One highly conserved pseudouridine synthase, TruD, catalyzes the formation of pseudouridine at position 13 of tRNA(Glu) in E.coli. TruD was the last pseudouridine synthase family to be discovered and shares the least global sequence homology with the other four known families. In addition, TruD does not have any homology to a known RNA binding motif and is likely binding its substrate via a novel mechanism. The main goal of this proposal is to characterize this novel and unique family of pseudouridine synthases. The proposal has been divided into two specific aims and proposes to answer questions about the mechanism and function of both the bacterial and eukaryotic members of the TruD family. The first aim is to examine the exquisite selectivity of the E. coli TruD homolog. Using standard biochemical means, including tritium release and filter binding assays, we will examine the features of the tRNA substrate and those of the enzyme, TruD, which modulate substrate recognition and binding. The second specific aim is to characterize the structure and function of TruD homologs of higher organisms. We will use X-ray crystallography to examine the structure of two human TruD homologs. The structural role of conserved sequence insertions found in TruD homologs of higher oganisms will be examined in the human homologs by crystallographic and biochemical means. RNA modification is important for RNA function, which in turn is essential for the maintenance of the cell's basic functions. Mutations resulting in a loss of pseudouridylation activity have been implicated in several human diseases. Studies proposed here will extend our understanding of the mechanism of a highly conserved class of RNA modification enzymes. These results are not only biomedically relevant, but will also serve to provide insight into the basic biology of cells.